Rickettsioses are good examples of diseases whose importance are not adequately appreciated despite their wide distribution throughout the world in the form of endemic foci, with sporadic and often seasonal outbreaks. Rickettsial infections have re-emerged in epidemic form in human populations, the epidemics of louse-borne typhus were responsible for over 30 million cases during and immediately after World War I, causing an estimated three million deaths. Despite the worldwide distribution of rickettsial diseases and the highly pathogenic nature of rickettsiae, there is a substantial gap in our understanding of the molecular mechanisms underlying rickettsial pathogenesis. Thus, this proposal is submitted to generate R. prowazekii, R. typhi, and R. rickettsii mutants lacking functional virulence genes and their inclusion in developing attenuated non-virulent strains for a broad-based protective rickettsial vaccines. To achieve this goal the following Specific Aims are proposed: (1) To clone and characterize genes encoding putative virulence proteins from R. typhi and R. rickettsii. Bioinformatic approaches will be utilized to identify genes that encode putative secreted proteins based on presence of the leader sequence;(2) To generate targeted gene replacement to develop rickettsial mutants that are attenuated for virulence. Experiments will be performed to disrupt virulence protein encoding genes of highly pathogenic rickettsiae (identified in Aim 1) to generate attenuated strains lacking virulent phenotypes as will be assessed by functional characterization of recombinant strains in vitro and in vivo;and (3) To characterize the recombinant rickettsia (from aim 2) for their immunogenicity and ability to protect against virulent rickettsial challenge in vivo, and to test the immunogenicity of recombinant proteins from aim 1 as potential vaccine candidates. The underlying hypothesis tested is whether recombinant strains with altered genes maintain their immunogenicity within mammals and provide cross protection to subsequent challenge with virulent strains.